Identification of Parthenogenesis-Inducing Effector Proteins in Wolbachia

Abstract Bacteria in the genus Wolbachia have evolved numerous strategies to manipulate arthropod sex, including the conversion of would-be male offspring to asexually reproducing females. This so-called “parthenogenesis induction” phenotype can be found in a number of Wolbachia strains that infect arthropods with haplodiploid sex determination systems, including parasitoid wasps. Despite the discovery of microbe-mediated parthenogenesis more than 30 yr ago, the underlying genetic mechanisms have remained elusive. We used a suite of genomic, computational, and molecular tools to identify and characterize two proteins that are uniquely found in parthenogenesis-inducing Wolbachia and have strong signatures of host-associated bacterial effector proteins. These putative parthenogenesis-inducing proteins have structural homology to eukaryotic protein domains including nucleoporins, the key insect sex determining factor Transformer, and a eukaryotic-like serine–threonine kinase with leucine-rich repeats. Furthermore, these proteins significantly impact eukaryotic cell biology in the model Saccharomyces cerevisiae. We suggest that these proteins are parthenogenesis-inducing factors and our results indicate that this would be made possible by a novel mechanism of bacterial-host interaction.


Introduction
The evolution of eukaryotic organisms has been continuously sculpted by relationships with intracellular microbes.The characteristics of these intracellular organisms have been driven by strong selection pressures to manipulate host physiology in favor of their own transmission and persistence (Moran et al. 2008).Many endosymbionts can be transmitted maternally but not paternally, an asymmetry that can result in sexual and genetic conflicts (Perlman et al. 2015).This phenomenon is exemplified by one of the most prevalent endosymbionts on earth, the bacterium Wolbachia.Wolbachia are maternally transmitted alphaproteobacteria (Rickettsiales) in many filarial worms and arthropods, including around half of all insect species (Kaur et al. 2021).Most remarkably, Wolbachia can drastically alter host reproductive outcomes in favor of their own transmission and spread, including the conversion of would-be male offspring to Wolbachia-transmitting genetic females, i.e. the induction of parthenogenesis.
Parthenogenesis induction (PI) is arguably the most advantageous reproductive phenotype for Wolbachia, as it is the only known symbiont-mediated reproductive manipulation in which host populations can be sustained with females only, resulting in all hosts being avenues for transmission of Wolbachia to the next generation.To date, PI-Wolbachia strains have been confirmed in a range of haplodiploid arthropods, including parasitoid wasps, thrips, and mites (Ma and Schwander 2017).Haplodiploidy is a sex determination system that relies on ploidy of the embryo: in the simplest form, males are haploid, and females are diploid.In many lineages, this difference in ploidy derives from whether a mother fertilizes a given egg, thus restoring diploidy (Gietz and Woods 2002; Fig. 1A).However, in the presence of PI-Wolbachia, diploid females can be produced from unfertilized eggs through a fertilization-independent, Wolbachia-mediated restoration of diploidy (Fig. 1B).
PI has evolved multiple times across Wolbachia as well as in several other genera of arthropod endosymbionts (Ma and Schwander 2017).Different occurrences of microbe-mediated PI are hypothesized to be made possible through different mechanisms, as inferred by cytogenetic approaches combined with estimates of heterozygosity (Stouthamer and Kazmer 1994;Gottlieb and Zchori-Fein 2001;Weeks and Breeuwer 2001;Pannebakker et al. 2004;Ma and Schwander 2017).For example, some PI-Wolbachia target meiosis to generate diploid gametes, while others target mitosis to convert haploid embryos to diploid.The PI-Wolbachia strains wTpre and wLcla cause a failed anaphase in the first mitotic division of the embryo leading to a single diploid nucleus in the parasitoids Trichogramma and Leptopilina, respectively (Fig. 1B;  Stouthamer and Kazmer 1994;Pannebakker et al. 2004), while the strain "wUni" causes gamete duplication through a tetraploid second mitotic prophase in a different parasitoid genus (Gottlieb and Zchori-Fein 2001), and "wBpra" induces functional apomixis in a species of mite (Weeks and Breeuwer 2001).Thus, PI bacteria have developed a variety of mechanisms for impacting host chromosome segregation.
In addition to differences in the timing and mechanisms for altering ploidy, there appears to be variation in whether a change in ploidy is sufficient to facilitate female development.In the wUni and wBpra systems, the diploidization event appears to be sufficient on its own to ensure female development.These have been termed "one-step PI" systems (Verhulst et al. 2023).In other PI systems, diploidization alone is not sufficient, as evidenced by the occasional presence of diploid phenotypic males, and even haploid and diploid individuals with intersex characteristics, especially when Wolbachia titers have been experimentally reduced (Tulgetske 2010;Lindsey and Stouthamer 2017).This implies a two-step mechanism of PI, wherein both diploidization and impacts on female-specific differentiation are required (Ma et al. 2015;Verhulst et al. 2023).The twostep PI mechanism agrees with mounting evidence suggesting that a number of PI-Wolbachia strains manipulate the host's gene expression cascade that is responsible for female development (Verhulst et al. 2023).Furthermore, in related sexual lineages, female development relies on a paternally derived factor (Fig. 1A), the absence of which could pose a constraint for the asexual reproduction of females (Zou et al. 2020;Verhulst et al. 2023).Across Hymenoptera, this sexual differentiation pathway centers around the gene transformer, the primary signal for female development (Verhulst et al. 2010).The sex determination cascade involves alternative splicing of transformer transcripts to exclude a premature stop codon to produce a functional, female-specific Tra protein, TraF.Male-specific splicing yields a nonfunctional "TraM" protein resulting in male-specific splicing of downstream transcripts, and therefore male development.In the parasitoid wasp Leptopilina clavipes, wasps infected with Wolbachia strain "wLcla" exhibit differential splicing compared to uninfected wasps, suggesting an interaction with the sex determination system, which could be the key to ensuring female development in such a two-step PI system (Geuverink et al. 2018).
Despite the discovery of microbe-mediated transitions to asexuality more than 30 yr ago (Stouthamer et al. 1990), the precise cellular mechanisms and bacterial genes responsible for manipulating host mitosis and reproductive biology have remained elusive.This is largely due to a paucity of genetic tools for Wolbachia, and, because PI-Wolbachia are not known to occur in genetically tractable models such as Drosophila, or even the model parasitoid Nasonia.Here, we focus on two-step PI-Wolbachia: wLcla infecting L. clavipes, and wTpre infecting Trichogramma pretiosum (Stouthamer and Kazmer 1994;Pannebakker et al. 2004).We hypothesized these Wolbachia would encode for an effector protein responsible for diploidization, and another responsible for driving female differentiation via interaction with the sex determination pathway.To test this hypothesis, we used comparative genomics across Wolbachia to identify two proteins, hereinafter termed PifA and PifB (for putative parthenogenesis-inducing factors A and B), and used molecular and functional assays to test their impacts on eukaryotic biology.Our results indicate that these are strong candidates as proteins responsible for the induction of parthenogenesis.

Results
Comparative Genomics Identifies Candidate Parthenogenesis-Inducing Factors (pifs) We identified 10 sets of orthologous proteins (a total of 11 individual proteins from wTpre and 20 from wLcla) that were both (1) shared between wLcla and wTpre and (2) absent from all other Wolbachia strains in our dataset.Twenty-seven of these genes (eight complete orthogroups) were pseudogenized (supplementary table S6, Supplementary Material online).The remaining two sets of orthologous proteins were putatively functional, and each set contained one hypothetical protein from wLcla and one from wTpre.For simplicity, these two sets of 1:1 orthologs are hereinafter referred to as PifA and PifB, for putative parthenogenesis-inducing factors A and B.
While the clustering of orthologous Wolbachia proteins identified only wTpre and wLcla as having PifA and PifB orthologs, we considered that (A) these proteins might be found in the published PI-Wolbachia strains that we did not include in our database (e.g. the one-step PI strain wUni; Gottlieb et al. 2002;Wang and Verhulst 2022), (B) there may be divergent Pifs present in other Wolbachia that did not meet clustering thresholds, (C) there might be unannotated pif-like regions present in other Wolbachia genomes, and (D) there may be Pif-like proteins in other organisms.To address some of these issues, we used blast to search for putative Pif homologs across nonredundant NCBI databases.Both protein and nucleic acid searches (blastp, tblastn, blastn, and discontiguous megablast) of pifA homologs only recovered matches to the already identified wTpre and wLcla PifA loci indicating that these are unique genes restricted to these PI-Wolbachia strains.Protein blast (blastp) searches of PifB identified short regions of distant homology to other proteins that contained leucine-rich repeat (LRR)-like domains.Outside of the wTpre and wLcla PifB orthologs, the highest scoring hit was a protein from an Alphaproteobacterium that aligned to 19% of the PifB sequence (at the LRR domains), with 30% amino acid identity (supplementary fig.S1 and table S11, Supplementary Material online).Lower scoring hits included a small number of proteins from other Wolbachia that had also acquired LRR-like domains, but, like the other database matches, they did not appear to be homologs of the wTpre and wLcla PifB proteins.Regions of similarity were divergent and restricted to the LRR-like domains, and the proteins did not globally align to the wTpre and wLcla Pifs or have any nucleotide-level sequence homology (supplementary fig.S1 and table S11, Supplementary Material online).
Both PifA and PifB have canonical features of eukaryotic host-associated bacterial effector proteins, including the aforementioned restricted phylogenetic patterns.Additionally, there are several shared characteristics with Wolbachia proteins that mediate other reproductive phenotypes.These similarities include proximity to phage and other mobile element loci, and location within a putative eukaryotic association module (EAM; Fig. 1C).The EAM is a region found in many Wolbachia genomes: it is associated with Wolbachia prophage (WO) regions or remnants, and in other Wolbachia strains the EAM contains loci responsible for cytoplasmic incompatibility and/or malekilling (Bordenstein and Bordenstein 2016;LePage et al. 2017;Lindsey et al. 2018;Perlmutter et al. 2019).Trichogramma-infecting Wolbachia were previously identified as having lost their WO phages (Gavotte et al. 2007), and genome sequencing of the wTpre strain revealed only degenerate prophage WO regions (Lindsey et al 2016).Indeed, the pif loci in wTpre are surrounded by those degenerate phage and transposable element loci, as well as other pseudogenes consistently found in the EAM, including mutL, a patatin-like gene, transcriptional regulators, and a suite of ankyrin domain containing proteins (Fig. 1C).The homologous pif loci in wLcla are contained within a short contig (wLcla_Contig_2; ∼11 kb) that also includes transposases, a transcriptional regulator, and pseudogenized hypothetical proteins.Due to the fragmented nature of the wLcla assembly, it is difficult to determine if the wLcla pif homologs are in a true prophage and/or EAM-like region.Finally, while pifA and pifB are syntenic in wTpre (329 bp between the 3′ end of pifA and the 5′ end of pifB), in wLcla the two genes are in opposite orientations and separated by a ∼1.6 kb region that contains a pseudogenized transposase and two small hypothetical proteins.
PifA and PifB contain eukaryotic-like domain structures: another key indicator of bacterial proteins that manipulate eukaryotic biology.Structural prediction revealed four regions of the Pif proteins with significant structural homology to eukaryotic domains (Fig. 2A; supplementary tables S7-S10, Supplementary Material online).The first is a domain with high similarity to nucleoporins at the N-terminus of PifA.Additionally, PifA contains two overlapping domains predicted to function in RNA splicing.Most notably, the second of these has significant structural similarity (33%) to the insect protein Transformer (Tra), discussed above for its role as the master-regulatory sex determination gene (Verhulst et al. 2010).Finally, PifB is primarily composed of a large LRR domain with strong structural homology to eukaryotic LRR receptor-like serine/ threonine protein kinases, especially membrane-bound Toll-like receptors (Fig. 2A).wTpre homologs of the Pif proteins were modeled with Robetta to visualize putative 3D protein structures (Fig. 2B and C; supplementary files S1-S4, Supplementary Material online).The predicted PifA protein structure consisted of a pore-like formation in the nucleoporin-like region (error estimate <10) and a relatively unstructured region aligning with the predicted RNA splicing/Tra-like domain.PifB structure results produced a double coiled structure typical of LRRs.Lastly, PifA and PifB are predicted to be bacterial secretion substrates, with a strong likelihood of secretion via the type IV secretion system, which is known to translocate Wolbachia effector proteins to the host in other strains (Whitaker et al. 2016) (supplementary table S12, Supplementary Material online).

Wolbachia Infection Is Correlated With Differential Splicing of Transformer and Double-Sex
Given the discovery of a Tra-like Wolbachia protein, and data indicating that Wolbachia strain wLcla impacts sexspecific splicing of tra in L. clavipes females (Geuverink et al. 2018), we queried previously published RNA-seq data from adult female T. pretiosum to assess the impacts of Wolbachia on tra and dsx splicing.These data are derived from three sets of wasp colonies, each with a genetically identical, Wolbachia-infected and -uninfected pair (n = 6 total).Exon use was previously assessed genome-wide, and here we display these patterns specifically for tra and dsx (Fig. 1D and E).We find that in female wasps without Wolbachia, exon 5 of tra and exons 3 and 5 of dsx are uniquely excluded.However, while female wasps with Wolbachia do use tra exon 5, there was no identifiable expression of tra exons 1 and 2.

pif Expression Correlates With Insect Reproduction
To see if pifA and pifB are expressed, and thus less likely to be an artifact of genome annotation, we used reverse transcription PCR (RT-PCR) to amplify pifA and pifB transcripts from a pool of ∼2-4-h old T. pretiosum embryos, and a pool of adult females.Indeed, both pifA and pifB are expressed in adult females, and we detected low or no expression of pifA and pifB in the young embryos (supplementary fig.S2, Supplementary Material online).Because pifA and pifB are syntenic in wTpre, reminiscent of the cytoplasmic incompatibility loci which are co-transcribed (Lindsey et al. 2018), we also used RT-PCR to look for the presence of pifA-pifB monocistronic transcripts, indicative of an operon.We were not able to amplify any monocistronic transcripts from adult females using primers that targeted the 5′ region of pifA and the 3′ region of pifB (supplementary fig.S3, Supplementary Material online).
To determine if pif expression correlated with reproductive biology, we used quantitative reverse transcription PCR (qRT-PCR) to quantify relative pifA and pifB expression across four time points in T. pretiosum development: early in embryogenesis, during the transition to metamorphosis, the "red-eye stage" of metamorphosis during which gonads undergo differentiation, and in adult females (Fig. 2D).There was a statistically significant increase in both pifA and pifB expression across wasp development (F 1,3 = 61.4,P < 0.0001).However, the relative expression of pifA was always significantly higher than pifB postembryonic development (F 1,3 = 18.51,P < 0.0001).At 2-4 h post-parasitization (shortly after the first mitotic division during which the Wolbachia-mediated gamete duplication step occurs; Stouthamer and Kazmer 1994;Pannebakker et al. 2004), there was minimal expression of pif genes.However, and importantly, these are measurements of mRNAs, so there may well be maternally loaded Pif proteins in these embryos.In freshly emerged adult females, expression of pifA and pifB was 4-and 1.5-fold higher than in embryos, respectively.Expression patterns of pifA and pifB in wLcla and L. clavipes recapitulated these patterns, though pifA had even greater expression than pifB (up to 25× higher; supplementary fig.S4, Supplementary Material online).

PifA Is Conserved and Repetitive in the Tra-Like Region
In validating plasmids and sequencing entry amplicons (see Methods), we discovered that the wTpre pifA gene (supplementary file S5, Supplementary Material online) contains a short region present in tandem triplicate that had been collapsed into a duplicate during genome assembly.The wLcla PifA ortholog was assembled correctly, based on our amplicon sequencing, though there were similar repeated regions in the same part of the coding sequence.Given these findings on the repetitive nature of PifA, we next characterized these patterns and their conservation.First, we compared the two orthologs to each other and we found a region of high similarity aligning with the Tra-like domain (Fig. 3A).Here, there is a ∼100 amino acid region of high synteny and conservation.This conserved region itself composed of tandemly repeated blocks of 12-18 amino acids (seen in the classic clustering of the dot plot in that region; Fig. 3A).To better visualize these tandem repeats, we compared each ortholog to itself (Fig. 3B and C).In wTpre, there are 4 of repeated segments in total, each 18 amino acids long (Fig. 3B).In wLcla, the repeated region was composed of 6 segments, each 12 amino acids long, and there was an additional instance of that repeated segment in the ∼250 amino acid region (Fig. 3C).

PifA and PifB Impact Eukaryotic Cells
To determine if PifA and PifB are putative effector proteins, we used a standard screen in the budding yeast, Saccharomyces cerevisiae.Bacterial effector proteins that interact with eukaryotic cell biology often cause growth defects when expressed in yeast cells (Kramer et al. 2007;Siggers and Lesser 2008;Sheehan et al. 2016;Beckmann et al. 2017;Rice et al. 2017).We expressed the wTpre PifA and PifB genes in W303 MATa yeast using a 2-μm expression vector with a galactose-inducible expression system, pAG424GAL-ccdB and/or pAG425GAL-ccdB (Alberti et al. 2007; Fig. 4).We additionally co-expressed PifA and PifB to test if there were synergistic or interactive effects on the yeast, which might indicate physical interaction between the two proteins, or impacts on the same structures or pathway.At 24 h, there were identifiable growth defects in the yeast expressing PifA or PifB, relative to the empty vector control.Co-expression of both Pifs also caused growth defects relative to the double vector control.We note that the yeast transformed with two high copy number 2-μm expression vectors generally do not grow as well, but there are still clear differences between the empty and Pif-containing conditions.By 48 h, there was still evidence for negative impacts on yeast growth in the PifA and co-expressing yeast.However, the yeast expressing only PifB were more similar to the empty vector controls at this later timepoint.

PifA Localizes With Atypically Dividing Nuclei
Finally, given the stronger growth impacts of PifA, and its nucleoporin-like domain, we used a fluorescent fusion tag (Green Fluorescent Protein; GFP) to localize PifA within the yeast cells.GFP alone displayed a homogeneous localization pattern spread evenly throughout the yeast cytoplasm (Fig. 5A).In contrast, GFP-PifA exhibited strong nuclear or perinuclear localization, reminiscent of localization patterns of known nucleolar proteins (Carmo-Fonseca et al. 2000;Taddei and Gasser 2012; Fig. 5B-D).Some yeast cells exhibited GFP-PifA puncti near each pole of dividing nuclei and deposition into the daughter cell (Fig. 5B and C).Other yeast cells had what looked like PifA dispersed around the dividing nucleus (Fig. 5D).Importantly, there was also evidence for atypical mitoses in the presence of PifA (Fig. 5D) with uneven distribution of daughter nuclei in the two cells.

Discussion
For decades the genes responsible for PI have remained elusive.This is despite increasing examples of microbemediated PI in nature, and significant progress made in understanding the mechanisms of other microbial reproductive manipulations such as cytoplasmic incompatibility and male-killing (Kaur et al. 2021).Indeed, PI is present in Wolbachia strains infecting at least six separate genera of parasitoid wasps (Ma and Schwander 2017).Because PI evolved multiple times across Wolbachia, we focused on strains predicted to share the same two-step mechanism of PI.As such, we identified and characterized two Wolbachia proteins that we predict are involved in the induction of parthenogenesis in T. pretiosum and L. clavipes: PifA and PifB.
Broadly, the proteins we identified have many characteristics of host-associated bacterial effector proteins.The pif genes have expression patterns that are coordinated with wasp development, with little to no expression in embryos, and high expression in adult females.We hypothesize there are maternally loaded Pifs present in embryos, and limited additional transcription happening, which correlates with the low levels of embryonic pif mRNA.Additionally, the short time window in which diploidization occurs likely necessitates pre-loading of the embryos with the appropriate Wolbachia effectors.Further support for the role of PifA and PifB as effector proteins includes growth phenotypes in yeast, and predictions that the Pifs are type IV secretion substrates (bacterial protein translocation systems that both wLcla and wTpre encode for; Lindsey 2020).We do note that yeast and insect mitoses have a number of key differences (e.g.open vs. closed; Sazer et al. 2014), and any signal indicating egg fertilization would likely not manifest in yeast cells, so it is unsurprising that the growth impacts in yeast are more mild than many other pathogenic bacterial effector proteins with broad host ranges (e.g. in Legionella; Siggers and Lesser 2008).
Regardless, the predicted domain structures of the Pif proteins are highly characteristic of functions in eukaryotic cells.PifA has both a nucleoporin-like region and an RNA-splicing-like region with homology to the insectspecific protein Transformer which is a strong indicator of interaction with host sex determination.The structural homologies of PifB to serine/threonine kinases, LRRs, F-box, and Toll-like receptor motifs indicate the ability to recognize, bind, and/or alter other proteins.LRR motifs are widespread in eukaryotic proteins and are typically involved in mediating protein-protein interactions (Kobe and Kajava 2001).Consequently, LRR motifs are also found in other bacterial effector proteins, acting to mimic and interface with host biology (Aepfelbacher et al. 2005;Xu et al. 2008).
In line with our hypotheses about the diversity of mechanisms underlying PI, there is no evidence for these proteins in other published Wolbachia genomes, including strains that use other PI mechanisms (e.g. the one-step PI FIG.4.-PifA and PifB impact eukaryotic cell biology.PifA and PifB cause growth defects in yeast relative to empty vector and no induction controls.Yeast strain W303 MATa was transformed with galactose-inducible 2-μm expression vectors that were either empty or contained a Pif coding sequence.Transformants were grown up in selective non-inducing media, normalized to OD600 = 1, and serial 1:10 dilutions were spotted on selective inducing or non-inducing plates and grown at 30 °C for 48 h.Single vector conditions leveraged the pAG425 backbone, and double expression conditions leveraged both pAG424 and pAG425 (see Supplementary Material online).
Identification of Parthenogenesis-Inducing Effector Proteins strain wUni; Wang and Verhulst 2022), based on blast results.Results from computational predictions for PifA and PifB also align with our expectation that two-step PI-Wolbachia need machinery for interacting with both chromosome division and the sex determination system.In two-step PI systems, the diploidization event during early embryogenesis is critical.This diploidization (in wTpre and wLcla, the failed anaphase) appears to be highly regulated as it only ever occurs during the first embryonic mitosis.If diploidization does not occur at the first mitosis (perhaps due to reduced Wolbachia titers), then it does not appear to happen at all (Tulgetske 2010).Additionally, PI-Wolbachia do not appear to cause rampant polyploidy during development, and Wolbachia-mediated diploidization does not occur in infected embryos if they have been fertilized (Stouthamer and Kazmer 1994).In many Trichogramma, PI-Wolbachia-infected females can mate and fertilize their eggs, in which case, the resulting offspring are diploid females and not triploid offspring (Stouthamer and Kazmer 1994), indicating that the PI mechanism is sensitive to both the specific mitotic event and to whether the egg has been fertilized.
Considering the specific conditions in which diploidization takes place, we hypothesize there is a host factor that serves as a signal for both the timing of the diploidization event (the first mitosis) and if diploidization is "not needed" (i.e. because the egg was fertilized).We argue it is unlikely for embryo ploidy itself to directly serve as this signal, for several reasons.First, in insects, maternal and paternal pronuclei stay physically separated for most of the first mitotic cycle (Tram et al. 2003).Second, even if Wolbachia or PI factors were capable of "counting chromosomes", a ploidy signal would not explain why the diploidization event only ever occurs at the first mitosis.Finally, interactions with a separate male sex-ratio distorter that is present in some Trichogramma species, the "paternal sexratio" (PSR) chromosome, indicate Wolbachia-mediated PI is sensitive to fertilization independent of ploidy.Specifically, the PSR "B-chromosome" is transmitted by haploid males: upon fertilization, the PSR chromosome mediates destruction of all other paternal chromosomes, resulting in a haploid male (a single maternal genome copy) plus the PSR chromosome which can then be transmitted to the next generation.Critically, when these PSR-males mate with Wolbachia-infected females, PSR-mediated distortions take precedence over Wolbachiamediated PI (Stouthamer et al. 2001).The paternal chromosomes are destroyed, leaving a haploid embryo with PSR, but Wolbachia-mediated diploidization never occurs.Thus, there is likely a non-chromosomal factor associated with fertilization, or its absence, that ultimately makes Wolbachia-mediated PI in this system possible both (1) during a specific mitotic event and (2) in the absence of fertilization.
A potential non-chromosomal factor that could signal both conditions is the presence or absence of paternally contributed centrioles.In fertilized hymenopteran embryos (e.g.Nasonia vitripennis), the sperm-tail basal body preferentially nucleates paternal centrioles to take part in the first mitotic division, and maternal cytoplasmic asters disperse (Ferree et al. 2008).In unfertilized, haploid embryos, this process is compensated for by de novo centrosome formation in the embryo from the maternal cytoplasmic asters (Ferree et al. 2006).The absence of a paternal centrosome in unfertilized eggs may serve as a signal or is possibly functionally significant in the PI mechanism.In the unfertilized embryo, if the PI proteins interact with the de novo formation of centrosomes, inhibition of proper chromosome segregation may result.Indeed, the centrosome looks to be a target of other sex-ratio modifying infections such as the male-killing bacterium Arsenophonus nasoniae in N. vitripennis.Here, the bacterium suppresses maternal centrosome formation in the unfertilized, male destined embryos.This results in mitotic spindle disruption, failed development of the early embryo, and ultimately malespecific mortality (Ferree et al. 2008).In this system, a PSR chromosome can also rescue the Arsenophonus malekilling phenotype, again pointing to potentially similar targets (Ferree et al. 2008).
Regardless of what the specific target of PI-Wolbachiamediated diploidization may be, we do know that an extensive network of protein kinases is required for successful mitosis in animal cells.These protein kinases, such as Aurora A, Aurora B, and polo kinase in Drosophila melanogaster, have multiple roles in regulating spindle assembly, centrosomal function, and cytokinesis (Archambault and Carmena 2012;Reinhardt and Yaffe 2013).The serine/ threonine protein kinase-like domain of PifB may indicate participation in these mitotic processes.Additionally, in many eukaryotes, mitotic phosphorylation sites typically lie in serine/threonine motifs (Canova and Molle 2014).Alternatively, PifA may be participating in mitotic disruption, as the N-terminal region has structural homology to a suite of nucleoporins that drive spindle assembly and chromosome organization in a wide range of other organisms (Chatel and Fahrenkrog 2011).Indeed, many of the PifA localization patterns in dividing yeast cells (Fig. 5) are reminiscent of the nucleoporin ALADIN in D. melanogaster (Carvalhal et al. 2015).ALADIN aggregates at the poles of dividing nuclei, and regulates localization of Aurora A (a serine/threonine kinase) and spindle organization (Carvalhal et al. 2015).There is also potential for two Pif proteins to interact to drive the failed first anaphase.For example, one protein's activity might be sensitive to the presence of the critical host factor (e.g.perhaps centrioles, as discussed above), and the other protein could then recruit the first protein to the critical host structures.This is of course speculation, but the localization patterns of PifA and mitotic impacts hint at more than interaction with the sex determination pathway.
In addition to this tightly regulated diploidization event, a second step is required to achieve female development in the wLcla and wTpre PI systems.In many sexual hymenopterans, female development seems to rely on a paternally derived signal.This might be in combination with a maternal effect (as in N. vitripennis; Beukeboom and Van De Zande 2010), or the paternal signal might be sufficient on its own (as is predicted to be the case in L. clavipes; Chen 2021).The absence of a paternal signal during asexual reproduction thus presents a constraint on female development.In the sexual species, N. vitripennis, the maternal tra allele is transcriptionally repressed in the embryo via an upstream factor, wom, and a combination of maternally loaded traF mRNAs and a transcriptionally active paternal tra allele is necessary for female development (Beukeboom and Van De Zande 2010;Zou et al. 2020).In contrast, maternally provided traF is absent in Wolbachia-uninfected L. clavipes embryos (Chen 2021), and it may be the case that paternal activation of the pathway or upstream factor that leads to traF is sufficient.Importantly, tra isoforms differ between Wolbachia-infected and Wolbachia-uninfected adult female L. clavipes.In the Wolbachia-infected wasps, only the traF variant is present, as compared to uninfected wasps where both traF and traM splice variants are present (Geuverink et al. 2018).Our data similarly indicate that Wolbachia is acting on the maternal pool of tra and dsx isoforms (Fig. 1D and E); this perhaps could be a result of the high expression of PifA at that timepoint in adult females (Fig. 2D).
The Tra-like domain within PifA may function as a replacement to initiate tra autoregulation and/or act on downstream sex determination genes such as dsx to ensure female differentiation.The nuclear localization of PifA in yeast cells notably complements its predicted nucleoporinlike region, suggesting that it may function within the host nucleus or perhaps on the nuclear membrane where posttranscriptional processing often occurs (Keene 2007).Indeed, previous experiments in T. pretiosum found that Wolbachia infection results in a number of differentially expressed and differentially spliced transcripts, and included in these are genes in the sex determination pathway (Wu et al. 2020; Fig. 1D and E).Given the predicted structure of PifA, these patterns could be attributed to direct splicing of host RNAs.However, it is also possible that PifA may act via allosteric or competitive interactions with the spliceosome, other RNA binding proteins, or other upstream processes.
PI microbes are clearly rich in factors for manipulating chromosome biology and sex determination of their hosts.Considering the impact of PifA and PifB on yeast biology, their location in a EAM genomic region, and structural similarities to eukaryotic domains involved in mitosis and sex determination, these proteins are promising candidates for PI.At present, we are quite limited by the lack of genetic tools in Wolbachia and natural hosts of PI-Wolbachia.Investigation of other Wolbachia phenotypes such as cytoplasmic incompatibility and male-killing has leveraged accessible heterologous expression systems in the D. melanogaster model: an insect that naturally harbors such symbionts.Indeed, tools in parasitic hymenopterans or other haplodiploid arthropods are quite sparse.Even in the most well-studied species, N. vitripennis, clustered regularly interspaced short palindromic repeats has only recently been used for gene-editing (Li et al. 2017;Chaverra-Rodriguez et al. 2020), but as of yet no one has achieved integration of a transgene in any Hymenopteran.Ongoing investigation into the mechanisms of symbiontmediated PI and deciphering the involvement of PifA and PifB will thus require a stronger focus on biochemical approaches and methods development for cell biology and genetics in these difficult to work with insects.Regardless, our study represents the first identification of putative parthenogenesis-inducing proteins, a major step toward understanding host-symbiont interactions and the evolution of asexual reproduction.

Genomics, Domain Prediction, and Protein Modeling
Thirty-three complete or near-complete Wolbachia genome assemblies representing nine supergroups (A, B, C, D, E, F, J, L, T) were used in comparative genomic analyses to identify putative parthenogenesis-inducing factors (supplementary table S1, Supplementary Material online).We specifically included the two PI-Wolbachia strains with a shared cytological mechanism for PI, wTpre and wLcla that infect the parasitoid wasps T. pretiosum and L. clavipes, respectively (Stouthamer and Kazmer 1994;Pannebakker et al. 2004).All genome sequences and prokaryotic genome annotation pipeline "RefSeq" annotations (Tatusova et al. 2016) were used to build orthologous groups of Wolbachia proteins using ProteinOrtho v5.15 with default parameters (Lechner et al. 2011).We identified groups of orthologous genes that were unique to the two PI-Wolbachia.Protein domain prediction was performed with HHpred (Söding et al. 2005;Zimmermann et al. 2018) using SMART_v6.0,PDB_mmCIF70_12_Oct, and Pfam-A_v34 as reference databases.Significant matches to structural domains were determined as those with probabilities greater than 50% or greater than 30% and in the top five hits, as per published HHpred best practices (Söding et al. 2005;Zimmermann et al. 2018).3D protein modeling was performed with Robetta's RoseTTAFold (Baek et al. 2021).Structures were visualized in PyMol (The PyMOL Molecular Graphics System, Version 2.0 Schrödinger, LLC).Secretion substrate prediction was performed with BastionX (Wang et al. 2021) and EffectiveDB (Eichinger et al. 2016).Dotplots were created in RStudio using the seqinr package, with parameters wsize = 20, wstep = 5, and nmatch = 5.Previously published RNA-seq data (Wu et al. 2020) were queried to visualize exon usage of transformer and double-sex transcripts in genetically matched female T. pretiosum with and without their Wolbachia infections (asexual and sexual, respectively).Supplementary table S2, Supplementary Material online, contains information on the annotations and accession numbers for the sex determining loci.
To compare the expression of Pifs across host development, we generated a synchronized developmental series for each wasp species.Adult T. pretiosum were given egg cards (E.kuehniella eggs adhered to cardstock with doublesided tape) for 2 h.Adult wasps were removed, and egg cards were held under standard rearing conditions until collection.Time points included: (1) 2-4 h post-parasitism, during the embryo stage, (2) three days post-parasitism, at the prepupal stage, when host Ephestia eggs turned gray, (3) seven days post-parasitism, at the red-eye pupal stage when ovaries are maturing, and (4) adult females less than 24 h post-eclosion that had not accessed fresh host eggs.Sampling times were based on published developmental studies of Trichogramma (Flanders 1937;Volkoff and Daumal 1994;Volkoff et al. 1995;Jarjees and Merritt 2002).Samples were collected in biological triplicate, and flash frozen by placement at −80 °C prior to further processing (see below).To prepare a synchronized developmental series of L. clavipes, one-day-old adult female wasps were given early second-instar D. virilis larvae and observed for parasitism.Parasitized fly larvae were left to develop either for (1) 2 h (embryo), (2) 14 days (early wasp pupation), or (3) 18 days (red-eye wasp pupae).Adult females were collected for gene expression analyses within 24 h of eclosion and prior to host access.Each stage was collected in biological triplicate as pools of three individuals, except for the embryo stage in which pools were composed of six individuals, to adjust for the small size.Samples were flash frozen and stored at −80 °C for further processing.
Nucleotide Extractions, PCR, RT-PCR, and qRT-PCR DNA was extracted from flash-frozen samples with the Monarch Genomic DNA Purification Kit (New England Biolabs), including the on-column RNase treatment.RNA was extracted from flash-frozen samples with the Monarch Total RNA Miniprep Kit (New England Biolabs) including the DNase treatment step.The LunaScript RT Master Mix Kit was used for making cDNA, following manufacturer's instructions.PCR reactions were performed with Q5 Hot Start High-Fidelity 2× Master Mix (New England Biolabs) in 20 µl reactions and products were run on a 1% agarose gel alongside an appropriately sized ladder (either New England Biolabs 100 bp or 1 kb DNA ladder) and stained post-electrophoresis with GelRed (Biotium).pif expression was assessed with the Luna Universal Two-Step RT-qPCR Master Mix (New England Biolabs) following manufacturer's instructions, and normalization to ftsZ (Wolbachia housekeeping gene).All reactions were run in technical duplicate alongside a standard curve and negative controls (including no template and no reverse transcriptase controls) on a QuantStudio 3 Real-Time PCR System (Applied Biosystems).All primer sequences are in supplementary table S3, Supplementary Material online.

Cloning and Yeast Transformation
To assess the impacts of Pifs on eukaryotic cells, we expressed them in the budding yeast S. cerevisiae.Coding sequences of pifA and pifB were PCR amplified from wTpre-infected wasps using modified forward primers to facilitate cloning into pENTR-D/TOPO (Invitrogen).These constructs were transformed into One Shot Top10 competent cells (Invitrogen) following manufacturer's instructions (see supplementary table S3, Supplementary Material online for primer sequences) and plated on selective media.Entry vectors were verified first with restriction digests, followed by whole-plasmid sequencing (Plasmidsaurus).Validated entry vectors were recombined into an appropriate destination vector (see supplementary table S4, Supplementary Material online) in an LR clonase reaction following manufacturer's instructions (Invitrogen).Expression vectors were cloned into One Shot Top10 competent cells (Invitrogen), plated on selective media, and validated as above.Yeast strain W303 MATa (supplementary table S5, Supplementary Material online) was transformed with expression vector(s) using the polyethylene glycol/Lithium acetate method (Yeast Transformation Kit, MilliporeSigma;Gietz and Woods 2002) and plated on non-inducing selective synthetic media (Sigma-Aldrich Yeast Synthetic Drop-Out Media Supplements).In all cases, plasmid extractions from Escherichia coli were performed with the Monarch Plasmid Miniprep Kit (New England Biolabs).Yeast transformants were grown overnight at 30 °C in selective noninducing media with 2% glucose as a carbon source.Cells were harvested by centrifugation and washed twice with sterile water, followed by resuspension in water to an OD 600 of 1.0.A five step, ten-fold dilution series was prepared for each transformant and 3 µl spots were plated on either selective inducing (2% galactose) or selective repressing (2% glucose) media.After spot plates were dried, they were incubated at 30 °C for 48 h.

Microscopy and Image Analysis
For fluorescence imaging, yeast transformed with GFP-tagged Pifs (or GFP alone) were grown overnight at 30 °C in selective non-inducing media, supplemented with 2% raffinose as a carbon source, and 20µg/ml of additional adenine to minimize autofluorescence (Rines et al. 2011).Cells were harvested via centrifugation and resuspended in selective inducing media (2% galactose) prior to incubation at 30 °C for an additional 6 h.After the 6-h induction period, yeast cells were stained with NucBlue Live ReadyProbes Reagent (Hoechst 33342; Invitrogen).Images were taken on an ECHO Revolve Fluorescence Microscope fitted with an 100 × Apochromat Oil Immersion lens, fluorescein isothiocyanate and 4',6-diamidino-2-phenylindole light emitting diode filter sets, and an extra long working distance Universal Condenser.Images were merged and pseudocolored in FIJI (Schindelin et al. 2012).

Statistics and Data Visualization
Statistical analysis and data visualization were performed in R 4.1.3(R Core Team 2014).Variation in pif expression was assessed with a two-way analysis of variance including gene and developmental stage as fixed effects, followed by post hoc testing with the Tukey-Kramer method.Schematics were created with BioRender.comand data visualization leveraged the "muted qualitative colour scheme" designed by Paul Tol (https://personal.sron.nl/∼pault/).

FIG. 1 .
FIG. 1.-Wolbachia-mediated parthenogenesis and putative PI factors.A) Haplodiploid sex determination in the absence of Wolbachia.Haploid males develop from unfertilized eggs, and diploid females develop from fertilized eggs.In some sexual hymenopterans, a paternal factor is required for driving female development in the offspring on top of fertilization (+pff).B) Parthenogenesis-inducing wTpre Wolbachia causes gamete duplication via a failed anaphase during the first mitotic division in Trichogramma embryos in the absence of fertilization.C) Candidate PI factors (pifs) are located within a degraded EAM.The schematic shows coding regions from base pairs 1,084,989 to 1,122,316 of the wTpre reference (National Center for Biotechnology Information (NCBI) Reference Sequence: NZ_CM003641.1;Lindsey et al 2016).D) Exon use of transformer and E) double-sex in female T. pretiosum with and without Wolbachia infections.Exons that are differentially skipped between Wolbachia-infected and -uninfected wasps are indicated with gray boxes.Schematics created with BioRender.com.Trichogramma photo taken by A.R.I.L.
FIG. 2.-Candidate parthenogenesis-inducing factors PifA and PifB.A) Domain prediction indicates PifA and PifB contain eukaryotic-like structures.Colored coordinates indicate amino acid positions of the relevant domain in the wTpre ortholog.B and B′) Two views of the predicted structure of the PifA protein from wTpre.B″ view is rotated 90° clockwise around the vertical axis as compared to the view in (B).C) Predicted structure of the PifB protein from wTpre.D) pif expression (relative to a bacterial housekeeping gene) increases across wasp development in T. pretiosum.